Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection

Veterinary Immunology and Immunopathology, 30 (1992) 233-246 Elsevier Science Publishers B.V., Amsterdam

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Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection* W.E. Owens, S.C. Nickerson and P.J. Washburn Hill Farm Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Rt. 1, Box 10, Homer, LA 71040, USA (Accepted 16 November 1990)

ABSTRACT Owens, W.E., Nickerson, S.C. and Washburn, P.J., 1992. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Vet. Immunol. Immunopathol., 30: 233-246. Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration, lntraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery ofS. aureus, but did not eliminate infections. Intravenous iujection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily. ABBREVIATIONS CFU, colony forming units; CNS, coagulase negative staphylococci; HBSS, Hank's balanced salt solution; MDF, milk derived factor.

INTRODUCTION

Recent research on bovine mastitis has focused on the possible uses of biological agents to modify the immune response of the mammary gland in*Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 90-80-5259.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$03.50

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creasing resistance to mastitis. Interleukin-2 has been investigated for its ability to enhance B-cell proliferation in cows, thus increasing resistance to Staphylococcus aureus mastitis (Baker, 1987; Nickerson et al., 1989b). Recombinant human granulocyte colony-stimulating factor has been evaluated for its ability to stimulate leukocytosis in milk and protect the mammary gland from experimental S. aureus challenge (Nickerson et al., 1989a). Endotoxin has been shown to induce a leukocytosis when infused into the mammary gland, resulting in protection from challenge with Streptococcus agalactiae (Brownlie, 1979) and reducing the severity of infection with S. aureus (Bramley et al., 1989). In addition to these biological agents, physical devices, including intramammary beads (Nickerson, 1987 ) and intramammary devices (Paape et al., 1981 ), have been shown to increase leukocyte numbers in milk and may increase resistance to mastitis pathogens. These agents and devices have an advantage when used in food animals because they are, for the most part, natural or inert products. This circumvents the problem of residues in milk, posed by more traditional mastitis therapies, such as antibiotics and anti-inflammatory steroids. In a previous study, pretreatment of mice with a novel oligosaccharide isolated from the milk of dairy cattle following hyperimmunization with a multivalent vaccine was shown to reduce mammary inflammation and involution of tissue after S. aureus challenge, and to increase the clearance of organisms when administered intraperitoneally (Owens and Nickerson, 1988 ). This milk-derived factor (MDF) is a low molecular weight (less than 10 000 Da) substance consisting primarily of carbohydrates with traces of 18-carbon fatty acids (Stolle Research and Development Corp., Lebanon, OH). This study describes the further evaluation of a more purified form of this compound in both cows and mice for anti-inflammatory properties and the ability to enhance the local immune response to S. aureus challenge in bovine and murine mammary glands. MATERIALS AND METHODS

Preparation o f M D F MDF was provided by Stolle Milk Biologics International and was prepared using their methods as described in U.S. patent number 4 956 394. Briefly, 20 1 of fresh milk from hyperimmunized cows were run through a cream separator to remove the fat. The resulting 16 1 of skimmed milk was ultrafiltered to remove molecular weight species greater than 10 000 Da using a hollow fiber diafiltration/concentrator (Amicon DL-10L). Twelve liters of the filtrate (less than 10 000 Da) was lyphilized yielding a dry powder. For further purification the dried power was reconstituted in double distilled water and applied to a sephadex G- 10 resin in a 2.5 × 80 cm glass column. The col-

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u m n was eluted at a flow rate of 30 m l / h . Fractions (3.3 ml) were collected and monitored at 254 n m and 280 nm. Three peaks were eluted, the M D F tested in these experiments appears in peak 1 as described previously in U.S. patent number 4 956 349. Cows were hyperimmunized using a heat-killed bacterial antigen preparation as described by Stolle Milk Biologics International in U.S. patent number 4 956 349. The bacterial antigens included: Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus pyogenes, A Type 1; Streptococcus pyogenes, A Type 3; Streptococcus pyogenes, A Type 5; Streptococcus pyogenes, A Type 8; Streptococcus pyogenes, A Type 12; Streptococcus pyogenes, A Type 14; Streptococcus pyogenes, A Type 18; Streptococcus pyogenes, A Type 22; Aerobacter aerogenes: Escherichia coli; Pseudomonas aeruginosa; Klebsiella pneumoniae; Salmonella typhimurium; Haemophilus influenza; Streptococcus mitis; Proteus vulgaris; Shigella dysenteriae; Diplococcus pneumoniae; Proprionibacter acnes; Actinomyces (anaerobe); Streptococcus mutans; Streptococcus sanguis ; Streptococcus salivarius and Streptococcus agalactiae. EFFECT OF MDF IN MICE

The effect of M D F on the LDso of S. aureus Newbould 305 for mice was determined using the m e t h o d of Reed and Muench ( 1938 ). Male CD- 1 mice were injected intraperitoneally with 10 or 100 m g / k g of M D F for 7 days prior to challenge. Three groups of six mice were challenged with 1.2 X 108, 1.2 X 109 or 1.2× 10 l° colony forming units ( C F U ) of the organism. Survival was determined at 24 h and the LDso calculated. The effects of intraperitoneal or intramammary injections of M D F on the murine m a m m a r y gland response to S. aureus challenge were determined in two groups of mice using the mouse mastitis model of Anderson ( 1977 ). For the group receiving intraperitoneal injections, gravid mice (five per group) were treated with 1, 10 or 100 m g / k g M D F for 3-4 days prior to parturition and 3-4 days after parturition for a total of 7 days. Peripheral blood was collected daily via puncture of the retro-orbital plexus. Total and differential leukocyte counts were determined and compared with values of untreated controls. After M D F treatment, treated and control mice were challenged intramammarily in the right (R4) and left (L4) upper abdominal m a m m a r y glands with 2.5 × 102 C F U S. aureus 305 in 0.1 ml saline. Twenty-four hours after challenge, mice were killed and the glands excised and examined bacteriologically and histologically. The other group of 10 mice was infused intramammarily with 1000/tg M D F in gland R4 3-5 days after parturition and 24 h prior to S. aureus challenge. Gland L4 served as a control and was infused with saline. Half of these mice were not challenged with S. aureus and were sacrificed by cervical dislocation 24 h after M D F infusion. Glands L4 and R4 were excised and processed for

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histology to determine the effect of M D F alone on m a m m a r y tissue. The remaining mice were challenged with S. aureus 24 h after M D F infusion. Twenty-four hours after challenge of both glands, mice were killed and the glands were removed aseptically. Glands were bisected, and one half processed for histology and the other half homogenized in 9 ml of sterile saline. The saline homogenates were plated to bovine blood agar to determine CFU ofS. aureus in M D F treated and control glands. Tissues from both groups of mice were used for histologic assessment and processed for light microscopic examination as described in Owens and Nickerson (1988). Briefly, tissue samples were fixed for 24 h in Bouin's fluid. Specimens (0.5-1.0 cm 3) were prepared for sectioning by infiltration and embedding in Paraplast (American Scientific Products, McGraw Park, IL). Tissue sections (5 /~m) were stained with hematoxylin and eosin and observed using a Zeiss standard 18 research microscope. The saline homogenate was plated to bovine blood agar to determine CFU of S. aureus per gland. Quantitative morphometric analysis was used to determine the percentage of m a m m a r y tissue area composed of alveolar epithelium, lumen and interalveolar connective tissue. For each tissue sample, 100 contact points were counted per slide at a magnification of 640 X. A reference grid in the microscope ocular provided fixed points used in the counting process. Tissue specimens of m a m m a r y parenchyma were also examined for the presence of leukocyte infiltration. Prevalence of these cells was quantified at 640 X in 10 randomly selected microscope fields per slide and assigned a score where 1 represents few or no leukocytes; 2 represents moderate leukocyte infiltration; and 3 represents intense leukocyte infiltration. Data were analyzed by analysis of variance using the general linear models procedure. Duncan's multiple range test was used to detect differences among means. Effects of treatment and dose were determined on the occurrence of the histologic and cellular components measured. Macrophage phagocytosis assay

Mammary gland macrophages were collected from a nonlactating Jersey cow by i n t r a m a m m a r y infusion of 50 ml of pyrogen-free saline followed by gentle massage. Lacteal secretions containing macrophages were collected into 20 ml of ice-cold Hank's balanced salt solution (HBSS) (Sigma Chemical Co., St. Louis, MO ) to a total volume of 50 ml. Cells were centrifuged, washed twice in ice-cold HBSS, and resuspended in 10 ml of HBSS. Cell concentration and viability were determined by counting a trypan blue stained preparation in a hemocytometer. Differential cell counts were determined by microscopic evaluation of Wright stained smears of the stock preparation. For the assay, cell concentration was adjusted to approximately l0 s macrophages/ml. The initial collection and wash resulted in a cell preparation containing 40-60% macrophages, with the balance being primarily lymphocytes.

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This preparation was dispensed in 1 ml aliquots to four-well tissue chamber slides (Lab Tek, Miles Scientific, Naperville, IL). Cells were allowed to adhere for 30 rain at 37 ° C. After adherence, wells were washed three times with warm HBSS (37 °C) to remove nonadherent cells, resulting in a preparation containing approximately 85% macrophages with the balance primarily lymphocytes. After the final wash, HBSS was replaced with Medium 199 (Sigma) supplemented with 0.68 m M glutamine and 25 m M Hepes (Sigma), and MDF was added to wells in concentrations ranging from 0.1 to 200/tg/ml. Cells were incubated with M D F for 24 h at 37°C with 5% CO2. After incubation, M e d i u m 199 was removed and cells were washed three times with HBSS. Opsonized S. aureus Newbould 305 (ATCC 29740) was added to each at a concentration of 105 C F U per well. Bacteria were opsonized by prior incubation with 10% normal bovine serum for 30 min. After 4 h incubation, cells were washed twice in HBSS and 5 u n i t s / m l of lysostaphin were added to lyse extracellular S. aureus. After 20 min incubation, cells were washed twice with HBSS and wells were treated with 0.05% saponin to lyse macrophages. The well contents were then aspirated and the number of viable S. aureus C F U / ml of lysate determined by standard plate count method on bovine blood agar. E f f e c t o f M D F in cows

The effect of i n t r a m a m m a r y infusion of M D F on the nonlactating bovine m a m m a r y gland response to S. a u r e u s challenge was determined as follows. Six cows in mid-dry period, each with at least two uninfected quarters, were infused daily in each uninfected quarter for 5 days with 50 ml sterile pyrogenfree saline. After each infusion, the quarters were gently massaged and milked out to enrich the lacteal macrophage population. On Day 6, one treated and one control quarter were infused with M D F (5 mg in 10 ml pyrogen-free saline) or pyrogen-free saline (10 ml), respectively. Twenty-four hours postinfusion, treated and control quarters were challenged by intracisternal infusion of 200 C F U S. a u r e u s 305. Secretion samples from all quarters were collected at 6 h post-challenge and daily thereafter for 5 days. Differential and total leukocyte counts were determined along with bacteriologic status. Isolation ofS. a u r e u s from two consecutive daily samples was considered confirmation of infection. The number of new S. a u r e u s infections in control and treated quarters and the differential and total somatic cell counts were compared. After initial treatment with M D F and challenge with S. aureus, quarters remaining infected with S. a u r e u s w e r e d i v i d e d into two groups with six infected quarters. One group was treated by daily infusion of M D F ( 5 mg) and the other was treated with pyrogen-free saline. The effect of M D F treatment on established S. a u r e u s infections was evaluated by determining the CFU recovered from S. a u r e u s - i n f e c t e d quarters and effect of treatment on differential and total m a m m a r y leukocyte counts.

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The effects of intravenous MDF injection on peripheral blood and mammary leukocyte profiles, as well as on existing S. aureus intramammary infections, were determined. Two cows in mid-dry period with a total of five S. aureus infected quarters were infused intravenously with 8 g of MDF suspended in 60 ml of pyrogen-free saline. Mammary secretion and blood samples were collected prior to treatment and at 24, 48 and 72 h post-treatment. Total and differential peripheral blood and mammary secretion leukocyte counts were determined. The S. aureus C F U / m l in quarter secretions was also determined at each sample time by plating aliquots to bovine blood agar. RESULTS

Effect o f M D F in mice

Daily intraperitoneal injection of 100 mg/kg MDF increased the LDso of 1 0 9 to more than 2 × 101° CFU. No effect on LDso was noted at the 10 mg/kg dose. Histologic and bacteriologic examination of mammary tissue from mice given daily intraperitoneal injections of MDF and challenged with S. aureus 305 indicated that all doses of MDF tested (1, 10 and 100 mg/kg) resulted in significantly greater percentage alveolar lumen, and significantly less stroma and leukocyte infiltration than control mice (Table 1 ). Thus, tissue from MDF-treated mice showed greater secretory activity and less inflammation compared with controls. MDF-treated glands also harbored fewer CFU S. aureus (approximately 2 X 105 ) than control glands (approximately 1 × 106 ); however, numbers were high in all groups. These same mice were also bled S. aureus 305 for mice from 5×

TABLE l Effect o f M D F intraperitoneal injection o n t h e leukocytic ~ a n d histologic 2 response o f m o u s e m a m m a r y glands 3 to i n t r a m a m m a r y challenge with 250 C F U Staphylococcus aureus 305 Treatment

Leukocytic infiltration Lumen Stroma Epithelium

HBSS

MDF ( 1 mg/kg)

MDF ( 10 m g / k g )

MDF ( 100 m g / k g )

2.3 a 19.4 a 37.8 a 42.8 ab

1.3 c 45.3 b 21.1 b 33.6 c

4.5 c 33.0 c 21.3 b 45.7"

1.5 c 37.7 ~ 22.3 b 40.0 b

~Data expressed as average score for infiltrating leukocytes where 1 represents very few or no neutrophils observed; 2 represents m o d e r a t e n e u t r o p h i l infiltration; a n d 3 represents m a r k e d neutrophil infiltration. "-Data expressed as percentage o f each m a m m a r y tissue c o m p o n e n t m e a s u r e d . 3Five mice per group. M e a n s w i t h o u t a superscript in c o m m o n differ ( P < 0.05 ).

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TABLE2 Effect of intramammary infusion of 1000/zg M D F on the leukocytic I and histologic2 response of mouse mammary glands 3just prior to challenge and 24 h post-challenge with 200 CFU Staphylococcus aureus 305

Leukocytic infiltration Epithelium Lumen Stroma

24 h prior to challenge

24 h post-challenge

Infused gland

Control gland

Infused gland

Control gland

2.9 a 28.7 55.4 15.9

1.0b 34.8 52.9 12.3

1.4 59.2 12.4 28.3

2.3 56.6 15.8 27.7

JData expressed as average score for infiltrating leukocytes where 1 represents very few or no neutrophils observed; 2 represents moderate neutrophil infiltration; and 3 represents marked neutrophil infiltration. 2Data expressed as percentage of each mammary tissue component measured. 3Five mice per group. Means without a superscript in common differ ( P < 0.05 ). TABLE 3 Effect of M D F on recovery o f Staphylococcus aureus 305 from mouse mammary glands after bacterial challenge by intramammary infusion I Mouse no.

1 2 3 4

CFU recovered Treated 2

Control 3

0 3.2X 103 0 0

3 . 6 × 105 3.1X103 4.0X 10 3 1.5X 10 4

t Mice were challenged intramammarily with 200 CFU Staphylococcus aureus 305 24 h after treatment with M D F or HBSS. 2Mice treated with 1000 #g M D F in the test gland. 3Control glands received HBSS.

daily during treatment with MDF. No effect on total and differential peripheral leukocyte counts was noted using any of the M D F doses compared with control mice. Infusion of 1000 #g M D F into mouse m a m m a r y # a n d s 24 h prior to S. a u r e u s challenge had a pronounced effect on the leukocyte response to infection and on the recovery of S. aureus. Glands infused with M D F and not challenged with S. a u r e u s had significantly more leukocytic infiltration (2.9 versus 1.0) by 24 h post-MDF infusion than # a n d s infused with HBSS indicating a mobilization of P M N (Table 2 ). MDF-treated glands challenged with S. a u r e u s exhibited less leukocytic infiltration (1.4 versus 2.3 ) 24 h post-

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challenge than HBSS-treated glands (Table 2 ). Glands receiving HBSS contained from 3.1 × 10 3 to 3.6 )< 10 5 CFU S. aureus per gland while three of four MDF-infused glands were free of bacteria (Table 3 ). One test gland showed no differences from controls; while reasons for this are uncertain, occasionally, the needle may not enter the lumen of the duct fully resulting in loss of the infusion into the subcutaneous tissue. This may have occurred in this gland. No difference was observed in the percent alveolar epithelium, lumen and connective tissue stroma between MDF-treated and control glands that did and did not receive bacterial challenge indicating no reduction of secretory potential caused by MDF infusion (Table 2 ). Effect o f M D F in cows

Incubation of bovine mammary macrophages with MDF resulted in increased phagocytosis of opsonized S. aureus. Macrophages incubated with 1000 or 2000/lg/ml MDF phagocytosed 1.04)< 103 and 1.43× 103 CFU of S. aureus, respectively, compared with 1.35 × 102 CFU phagocytosed by control macrophages, indicating an enhancement of phagocytosis by MDF. Intramammary infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer quarters becoming infected (five of ten ) than saline-treated control quarters (seven of nine ). In those quarters becoming infected, the number of S. aureus being shed was lower in MDF-treated quarters than in saline control quarters the first 3 days (Table 4 ). The percent of neutrophils was higher in MDF-infused quarters at the time of S. aureus challenge (mean 78%) than in saline-infused quarters (mean 45%) but levels were similar by 48 h post challenge (Fig. 1 ). Repeated daily infusion of S. aureus infected and uninfected quarters of the above cows again increased the percent neutrophils in mammary secretions to over 80% by 5 h post MDF infusion (Fig. 2). The mean C F U / m l of S. aureus shed from infected quarters decreased initially by 5 h apparently as TABLE4 Mean CFU Staphylococcus aureus/ml of milk from infected quarters of cows pretreated with MDF or saline and challenged with 200 CFU Staphylococcus aureus Time post challenge

MDF treated ( n = 10)

Saline treated (n=9)

Oh 6h I day 2 days 3 days 5days

0 475 8400 (4) t 43 000 (4) 56 700 (5) >100000(5)

0 9850 (4) 16 700 (7) 20 600 (7) 89 000 (7) >100000(7)

qndicates number of infected quarters.

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EFFECT OF MILK-DERIVED FACTOR ON INFECTION 100

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~

UNINFECTED* SALINE

* COWS CHALLENGED WITH S. AUREUS AT 24HR

Fig. 1.Percent neutrophils in secretions from quarters of cows infused intramammarily with 5 mg MDF or pyrogen-free saline. Ten MDF-infused quarters and nine saline-infused quarters were subsequently challenged with 200 CFU S. aureus at 24 h. 100 80 P E R C E N T

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Fig. 2. Percent neutrophils in secretions from quarters of cows retreated daily with 5 mg MDF or pyrogen-free saline. a result o f M D F i n f u s i o n . H o w e v e r , C F U / m l i n c r e a s e d steadily a f t e r 24 h d e s p i t e daily M D F i n f u s i o n . I n t r a v e n o u s i n j e c t i o n o f 8 g M D F h a d a p r o n o u n c e d effect o n the differe n t i a l a n d t o t a l l e u k o c y t e c o u n t o f the t w o test cows. T o t a l l e u k o c y t e c o u n t s d r o p p e d f r o m 8 0 0 0 / m m 3 to 1000 l e u k o c y t e s / m m 3 w i t h i n 1 h (Fig. 3). By

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Fig. 3. Effect of intravenous injection of 8 g MDF in 60 ml pyrogen-free saline on the total peripheral leukocyte count of two cows. Fig. 4. Effect of intravenous injections of 8 g MDF in 60 ml pyrogen-free saline on the mammary leukocyte count (SCC) from all quarters of two cows. Fig. 5. Effect of intravenous injection of 8 g MDF suspended in 60 ml pyrogen-free saline on the mammary leukocyte count (SCC) from S. aureus-infected quarters (n = 5) of cows injected with MDF. Fig. 6. Effect of intravenous injection of 8 g MDF suspended in 60 ml pyrogen-free saline on the mammary leukocyte count (SCC) from CNSinfected quarters ( n = 3 ) of two cows injected with MDF.

rn

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24 h, counts had returned to normal ( 7 0 0 0 / m m 3) and continued to rise to a m a x i m u m of 14 0 0 0 / m m 3 by 72 h. Percent neutrophils dropped from 37% to less than 1% within 1 h and began to increase rapidly, rising to 12% by 4 h and 70% by 24 h. Thus, systemic M D F injection caused a severe transient leukopenia within 1 h post-injection followed by a transient compensatory increase to concentrations above pretreatment values. The accompanying M D F effect on m a m m a r y leukocytes across all quarters regardless of infection status was less dramatic but followed a similar time course. Total leukocyte count dropped from 11 370X 103 at time 0 to 6250X 103 at 4 h, peaked at 27 7 6 0 × 103 at 24 h, and dropped to 4980 at 72 h (Fig. 4). The effect was much more pronounced in S. a u r e u s infected quarters (Fig. 5 ) than in quarters infected with coagulase-negative staphylococci (CNS) (Fig. 6). The n u m b e r of bacteria recovered from S. a u r e u s and CNS-infected quarters decreased somewhat from 0 to 4 h after M D F injection but all quarters remained positive for bacteria at all sample times. M D F was shown by limulus lysate assay to contain a low concentration of endotoxin (approximately 10-100 n g / m g ) . Therefore, in the 5 mg M D F dose that was infused, endotoxin contamination was 50-500 ng. To ensure that any effects of M D F observed were not caused by endotoxin, two control experiments were performed. In the first, a Jersey heifer was infused intramammarily into two quarters with 100 or 1000 ng of endotoxin (twice the range found in M D F ) to determine the effect on differential leukocyte counts. No effect on percent neutrophils was observed by infusion of these levels of endotoxin. In the second control experiment, two dry cows were infused in each quarter with 250 ng endotoxin and challenged in all four quarters with 200 C F U S. a u r e u s 305 24 h after endotoxin infusion. The two cows had a total of seven quarters eligible for S. a u r e u s infection. Results showed that endotoxin infusion had little effect on percent neutrophils in secretions from these two cows. Also, six of the seven eligible quarters became infected indicating no protection by this dose of endotoxin. Thus, the effects noted previously were attributed to M D F and not to endotoxin contamination. DISCUSSION M D F appeared to exhibit a nonspecific enhancement of host defenses against bacterial challenge as well as anti-inflammatory activity depending upon route of administration. Results of the mouse experiments were in agreement with a previous investigation with this c o m p o u n d in a less purified form (Owens and Nickerson, 1988 ). M D F injection increased the n u m b e r of S. a u r e u s necessary for lethality suggesting some protective effect. M a m m a r y glands of mice pretreated via daily intraperitoneal injection of M D F were less

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inflamed (as evidenced by reduced leukocytosis) than glands of mice receiving HBSS after bacterial challenge. Conversely, direct infusion of MDF into mouse mammary glands was inflammatory, but resulted in substantial protection against subsequent intramammary challenge with S. a u r e u s compared with uninfected controls. The nature of this protection is uncertain; however, the nonspecific inflammation induced may have been responsible in part for the protection against bacterial challenge. Similarly, Bramley et al. (1989) demonstrated that stimulation of an inflammatory response in the mouse mammary gland using endotoxin reduced recovery of S. aureus after subsequent challenge. Bovine macrophages incubated with MDF phagocytosed more opsonized S. aureus than did macrophages incubated with HBSS indicating that MDF possesses modulatory properties for macrophages. This enhancement of phagocytosis may explain in part the protective effects of MDF observed in mice and cows treated with MDF prior to bacterial challenge. A similar stimulation of mouse macrophages has been observed by researchers for glycans (Buddle et al., 1988; Selgelid et al., 1981 ). For example, glucan, a fl-l,3 polyglucose derived from yeast, was shown to enhance humoral and cellular immunity via macrophage modulation. More recently, Newburg et al. (1989) reported protection from E s c h e r i c h i a coli stable toxin in a suckling mouse model by a fucosyloligosaccharide isolated from human milk. No effect on peripheral leukocyte counts was observed in mice after intraperitoneal injections of MDF. However, intravenous injection of 8 g MDF in cows resulted in a rapid and dramatic leukopenia followed by a subsequent rise in total leukocyte count to above pretreatment levels before a return to normal levels. Mammary leukocyte cell counts were also affected by intravenous injection of MDF. The effect was much more pronounced in quarters harboring S. aureus infections than in those infected with CNS. The reason for this marked difference in response is uncertain. S t a p h y l o c o c c u s aureus infected quarters often have more tissue damage than do those harboring the less pathogenic staphylococcal species, and as a result, mammary tissues have more interaction with peripheral blood and serum. Milk from cows with infected quarters have increased concentrations of serum components such as immunoglobulin, serum albumin, sodium and chloride (Eberhart et al., 1987 ); also, S. a u r e u s infected quarters typically achieve higher antibiotic levels in tissue from systemically administered antibiotic caused by the break down of the blood-mammary gland barrier as a result of infection (Owens and Nickerson, 1990). Therefore, the enhanced response to systemic MDF in infected quarters may have occurred because of increased exposure of mammary tissue to MDF as a result of infection. MDF infusion into uninfected quarters resulted in increased mammary leukocyte counts and a corresponding partial protection from infection after intramammary challenge with 200 CFU S. aureus. In addition to increased

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resistance to infection, those M D F - i n f u s e d quarters b e c o m i n g infected shed fewer bacteria t h a n u n i n f u s e d control quarters. Daily infusion o f M D F into S. a u r e u s - i n f e c t e d quarters r e d u c e d the severity o f infection as e v i d e n c e d by a decrease in total bacteria being shed f r o m those quarters, but the p r o t e c t i o n was not sufficient to effect a cure. Perhaps a c o m b i n a t i o n o f M D F t r e a t m e n t with antibiotic t h e r a p y would have a u g m e n t e d the benefits o f bot h and increased chances for cure. Thus, M D F exhibited significant biologic activity in mice and cows, markedly affecting leukocyte n u m b e r s when a d m i n i s t e r e d systemically in cows. Some a n t i - i n f l a m m a t o r y activity and p r o t e c t i o n from, or a m e l i o r a t i o n of, infection was n o t e d but this did not a ppe ar to be sufficient to be t herapeut i c alone. ACKNOWLEDGEMENTS T h e authors t h a n k C o r i n n e R a y a nd N a n c y Boddie for technical assistance, Frances C. H u f f for typing o f the manuscript, and Fort Dodge Laboratories for partial s uppor t o f this study.

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